Fecal pollution is a persistent problem, affecting many coastal and inland waters in the United States. Despite improvements in wastewater treatment technology and management practices, fecal contamination from many sources finds its way into our waters, jeopardizing the health of the ecosystems and everything that depends on them. The problem continues partly because current methods are unable to identify the source. Fecal contamination may be introduced into natural waters from a variety of sources, including ineffective sewage treatment, leaking septic systems, illegal dumping, recreational boaters, agricultural runoff, and wildlife.
Point sources, such as sewage treatment plants, can be monitored easily, and appropriate actions can be taken to reduce or eliminate the discharge. Conversely, non-point sources, such as runoff from urban and rural areas, are much more difficult to trace, and often constitute a significant portion of the contamination. Prior to the 1970's, pollution from agriculture and wildlife was considered “natural and uncontrollable” (Martin, J. Environ. Qual. 26:1198–203, 1997). However, several high-profile incidents involving fish kills and shellfish bed closures increased public awareness, and in 1971, the United States Army Corps of Engineers began issuing permits for discharging wastes into navigable streams and their tributaries (Id.). Since then, numerous studies have investigated the effects of agricultural runoff on bacterial pollution in natural waters (Patni et al., Transactions of the American Society of Agricultural Engineers 28:1871–84, 1985; Fernandez-Alvarez et al., J. Appl. Bact. 70:437–42, 1991; Niemi et al., J. Environ. Qual. 20:620–7, 1991; Miner et al., “Fecal Coliform Concentrations Measured at Various Location in the Tillamook Watershed 1965–1993”, Post Project Analysis of the Tillamook Rural Clean Water Project, Preliminary Report No. 1, Bioresource Engineering Department, Oregon State University, Corvallis, Oreg., 1994; Edwards et al., J. Amer. Water Resources Assoc. 33:413–22, 1997; Stoddard et al., J. Environ. Qual. 27:1516–23, 1998; and Baudart et al., J. Environ. Qual. 29:241–250, 2000). Runoff from agricultural land is now considered one of the major sources of non-point pollution.
One of the more obvious ways that fecal pollution interferes with human activities is the introduction of pathogens commonly found in feces. Bacterial and other pathogens include organisms such as Salmonella, Shigella, E. coli O157:H7, Cryptosporidium, and Giardia. Viruses such as Hepatitis, Norwalk, and other enteroviruses also are often associated with feces and are detected commonly in environmental water samples (Havelaar et al., Appl. Environ. Microbiol. 59:2956–62, 1993; Paul et al., Appl. Environ. Microbiol. 63:133–8, 1997; Griffin et al., Appl. Environ. Microbiol. 65:4118–25, 1999; and Vantarakis et al., Water, Air, and Soil Pollution 114:85–93, 1999). Many of these pathogens survive in the environment (Rhodes et al., Appl. Environ. Microbiol. 45:1870–1876, 1983; Sinton et al., Appl. Environ. Microbiol. 60:2040–8, 1994; Mezrioui et al., Water Res. 29:459–465, 1995; Bosch et al., Water Science and Technology 35:243–7, 1997; Murrin et al., Water Science and Technology 35:429–32, 1997; Pallin et al., J. Virol. Meth. 67:57–67, 1997; Sinton et al., Appl. Environ. Microbiol. 65:3605–13, 1999), and thus are cause for concern.
In addition to pathogens carried in feces, large amounts of nutrient-rich material are introduced into receiving waters. This sudden surge of nutrients, such as nitrogen, phosphorus, and organic carbon, can cause severe imbalances in the ecosystem. For example, Cloern and Oremland (Estuaries 6:399–406, 1983) found increases in decomposition and nitrification, leading to anoxia, following the discharge of a large volume of primary-treated sewage into San Francisco Bay. Other studies have focused on the effects of nutrient enrichment from agricultural runoff. Phosphorus found in runoff from agricultural land was shown to increase as the proportion of land used in agricultural practices, such as dairy waste application, increased (McFarland et al., J. Environ. Qual. 28:836–844, 1999). This is significant because of the potential for eutrophication in phosphorus-limited freshwaters (Daniel et al., J. Environ. Qual. 27:251–7, 1998). Additionally, allochthonous material from non-point sources can induce alga1 blooms, some of which may be harmful (see Paerl, Limnology & Oceanography 33:823–47, 1988 for review).
Because of these imminent threats, fecal pollution is frequently monitored in many coastal waters, especially those areas used for shellfisheries and recreation. The most commonly used measure of fecal pollution are the number of viable coliforms, fecal coliforms, or Escherichia coli in a water sample (American Public Health Association, Standard Methods for the Examination of Water and Wastewater, 18th edition, Washington, D.C., American Public Health Association, 1992). Fecal coliforms are defined as gram negative, non-sporulating, rod-shaped bacteria that ferment lactose with gas formation within 24 hours at 44.5° C. (Id). They have been used as indicator bacteria for many years and are currently the Environmental Protection Agency standard for assessing water quality.
Despite the wide use of fecal coliform tests, many researchers and water resource managers have begun to question the validity of these measurements (see Toranzos et al., Environ. Toxicol. Water Qual. 6:121–30, 1991 for review). Although these bacteria typically originate from fecal material, fecal coliforms can survive and grow outside of enteric habitats (Flint, J. Appl. Bact. 63:261–70, 1987; Alkan et al., Water Res. 29:2071–81, 1995; Davies et al., Appl. Environ. Microbiol. 61:1888–96, 1995; Mezrioui et al., Water Res. 29:459–65, 1995; Bogosian et al., Appl. Environ. Micro. 62:4114–20, 1996; and Stoddard et al., J. Environ. Qual. 27:1516–23, 1998). They often settle in sediments, where they can grow, and then be resuspended during mixing events (Sherer et al., Transactions of the American Society of Agricultural Engineers 31:1217–22, 1988; Sherer et al., J. Environ. Qual. 21:591–5, 1992; and Davies et al., Appl. Environ. Microbiol. 61:1888–96, 1995). Thus, measurements of fecal coliforms may not accurately reflect recent contamination.
Ribosomal RNA is a direct gene product and is coded for by the rRNA gene. The DNA sequence for rRNA is used as a template to synthesize rRNA molecules. A separate gene exists for each of the ribosomal RNA subunits. Multiple rRNA genes exist in most organisms, higher organisms containing both nuclear and mitochondrial rRNA genes. Plants and certain other forms contain nuclear, mitochondrial, and chloroplast rRNA genes.
Numerous ribosomes are present in all cells of all life forms. About 85–90 percent of the total RNA in a typical cell is rRNA. A bacterium such as E. coli contains about 104 ribosomes per cell while a mammalian liver cell contains about 5×106 ribosomes. Since each ribosome contains one of each rRNA subunit, the bacterial cell and mammalian cell contains 104 and 5×106, respectively, of each rRNA subunit.
Nucleic acid hybridization, a procedure well known in the art, has been used to specifically detect extremely small or large quantities of a particular nucleic acid sequence, even in the presence of a very large excess of non-related sequences. Prior uses of nucleic acid hybridization are found, for example, in publications involving molecular genetics of cells and viruses, genetic expression of cells and viruses, genetic analysis of life forms, evolution and taxonomy of organisms and nucleic acid sequences, molecular mechanisms of disease processes, and diagnostic methods for specific purposes, including the detection of viruses and bacteria in cells and organisms.
Polymerase chain reaction, or PCR, produces many copies of a particular template DNA sequence in vitro. This process, well known in the art, uses nucleic acid hybridization to hybridize two primers, consisting of short DNA oligonucleotide molecules, to complementary sites on either side of the DNA sequence to be copied, or amplified. The use of a thermally-stable DNA polymerase allows repeated cycles of template denaturation, primer annealing, and synthesis of the template sequence. Specificity of the reaction is controlled by the primer design and the reaction conditions. Prior uses of PCR are found, for example, in publications involving studies of genetics of cells and viruses, genetic expression within cells, evolution and systematics of organisms, and diagnostic applications in clinical, industrial, and other settings.